[Show abstract][Hide abstract]ABSTRACT: Type 2 diabetes is caused by insulin resistance coupled with an inability to produce enough insulin to control blood glucose, and thiazolidinediones (TZDs) are the only current antidiabetic agents that function primarily by increasing insulin sensitivity. However, despite clear benefits in glycemic control, this class of drugs has recently fallen into disuse due to concerns over side effects and adverse events. Here we review the clinical data and attempt to balance the benefits and risks of TZD therapy. We also examine potential mechanisms of action for the beneficial and harmful effects of TZDs, mainly via agonism of the nuclear receptor PPARγ. Based on critical appraisal of both preclinical and clinical studies, we discuss the prospect of harnessing the insulin sensitizing effects of PPARγ for more effective, safe, and potentially personalized treatments of type 2 diabetes.

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between the three TZDs. Approximately 25% of the IRdsubjects didnotrespondtoTZDtreatment,demonstratingminimalornoimprove-ment in insulin-sensitivity. To contrast the expression profiles of IRdTZD-responders (those having the best insulin sensitization response)with IRdTZD-nonresponders (those having the worst insulin sensiti-zation response), all of the IRdsubjects (referred to below as IRdall)were ranked by their fractional Rdchange and divided into quartilesubgroups. IRdsubjects in the top quartile of fractional change in Rd(?83%increaseinRd)wereclassifiedasIRdresponders(IRd?).IRd?subjects not only had the largest fractional Rdchange but also had thegreatestimprovementinplasmainsulinlevels(DatasetS1).IRdsubjectsin the bottom quartile of fractional Rdchange (?13% increase in Rd)wereclassifiedasIRdnonresponders(IRd?).IRdsubjectsinthemiddletwo quartiles of fractional Rdchange (13–83% increase in Rd) wereclassifiedasIRdmidresponders(IRdmid).Fig.1BshowsthedistributionoftheseIRdsubgroups.Thus,theIRdallsubjectgroupreferstothesumof the IRd?, IRd?, IRdmidsubject groups.Insulin Resistance Is Associated with Impaired Insulin-Induced MetabolicGeneExpressionChangesinSkeletalMuscle,WhichAreLargelyNormalizedAfterTZDTreatment.WeanalyzedSMgeneexpressionprofilesfromthesubjectgroupsandidentified?150geneswhosepre-TZD,basaland/orpostclampexpressionlevelsweresignificantlydifferentinIRdallsubjects(?Bonf?0.05)comparedwithNRdsubjects(DatasetS2).Amongthesegeneswerecriticalregulatorsoflipidmetabolism.Forexample,expres-sion of lipoprotein lipase (LPL), a critical regulator of SM lipidmetabolism (11) and a target gene of PPAR? (12), was 48% lower inIRdallsubjects in the pre-TZD fasting state (?Bonf? 0.05), was fullyrescued by TZD treatment, and was positively correlated with Rd(r ?0.53). All genes with baseline expression values that correlate with RdareshowninDatasetS3.Wealsoidentified237insulin-targetgenes,i.e.,genes suppressed or activated in the insulin-stimulated (postclamp)state in NRdsubjects. One hundred sixteen insulin-target genes werefunctionally involved with metabolism (Dataset S2). The insulin-induced response of 42 genes was significantly altered in the IRdallsubjects,ofwhich19geneswerefunctionallyinvolvedwithmetabolism.IRdallsubjectsexhibitedimpairedinsulin-inducedexpressionchangesinhexokinase 2 (HK2) and pyruvate dehydrogenase kinase 4 (PDK4)compared with NRdsubjects. HK2 expression is normally induced byinsulin, but this induction was blunted in IRdallsubjects (Fig. 2A).Consequently,thepostclampHK2expressionlevelinIRdallsubjectswas50% lower (?Bonf? 0.05) than in NRdsubjects. Insulin activation ofHK2 commits glucose to the intracellular compartment in muscle,facilitating glucose metabolism in the fed state. PDK4 expression isnormally suppressed by insulin, but this effect was blunted in IRdallsubjects(Fig.2B).Asaresult,thepostclampPDK4expressionlevelinIRdallsubjectswas7-foldhigherthaninNRdsubjects.PDK4,initsactivestate,inhibitspyruvatedehydrogenasecomplexconversionofpyruvateinto acetyl-CoA, effectively disrupting glucose utilization. The sche-maticinFig.2Cshowshowdefectiveinsulin-inducedregulationofHK2and PDK4 in insulin resistant subjects would reduce glycolytic flux.Additional genes involved in the glycolytic pathway (PFKFB3 andPKM2)exhibiteddefectiveinsulin-inducedregulationinIRdallsubjects,also shown in Fig. 2C. We observed insulin-induced, transcriptionallycoordinatedrepressionoftheinsulinsignalingpathway(Fig.S1).AfterTZD treatment, IRdallsubjects exhibited improved insulin-inducedHK2 and PDK4 expression changes and postclamp HK2 and PDK4expression levels compared with NRdsubjects (red and blue bars,respectively, in Fig. 2 A and B). We contrasted these HK2 and PDK4expression patterns with those from the responder (IRd?) and nonre-sponder(IRd?)subgroups(pinkandyellowbars,respectively,inFig.2A and B). Interestingly, post-TZD improvements in the expression ofHK2 and PDK4 were more robust in the IRd?subject subgroupcompared with the IRdallsubject group and absent in the IRd?subjectsubgroup (Fig. 2 A and B). Thus, TZD-improved insulin-regulation ofHK2andPDK4,andpresumablyglycolyticflux,isrelatedtothedegreeof TZD-improved insulin sensitization.Fig. 1.human insulin-resistance study. (B) Distribution of baseline insulin sensitivity andTZD-mediated insulin sensitization response of study subjects. NRd(insulin sensitivesubjects with normal Rd, ?8 mg/kg per min, blue square symbols), IRdall(insulinresistantsubjectswithimpairedRd,?8mg/kgpermin,trianglesymbols),IRd?(sub-group of insulin resistant subjects that are TZD responders, in upper quartile ofpost-TZD fractional Rdchange, pink triangles), IRd?(subgroup of insulin resistantsubjects that are TZD nonresponders, in bottom quartile of post-TZD fractional Rdchange,yellowtriangles),IRdmid(subgroupofinsulinresistantsubjectsinthemiddletwoquartilesofpost-TZDfractionalRdchange,redtriangles).RdFrac?fractionalRdchange[(Rd2?Rd1)/Rd1].Study design and subject insulin sensitivity distribution. (A) Schematic ofFig. 2.and B) Pre- and postclamp HK2 and PDK4 expression data from the subject groupsbeforeandafterTZDtreatmentareshown.(A)Insulin-activatedexpressionofHK2inNRdsubjects was completely blocked in IRdallsubjects. After TZD-treatment, thisdefect was improved in IRdallsubjects, normalized in IRd?subgroup subjects andunaffectedinIRd?subgroupsubjects.(B)Insulin-inducedrepressionofPDK4expres-sioninNRdsubjectswasbluntedinIRdallsubjects.AfterTZD-treatment,thisdefectwasimproved in IRdallsubjects, normalized in IRd?subgroup subjects and unaffected inIRd?subgroup subjects. NRd(blue), IRdall(red), IRd?subgroup (pink), IRd?subgroup(yellow).(C)Glycolysisschematichighlightingdefectivelyregulatedgenesbyinsulinin IRdallsubjects compared with NRdsubjects before TZD treatment. Green fill indi-cates significantly blunted insulin-induced expression in IRdallsubjects, resulting inlow postclamp expression. Red fill indicates significantly blunted insulin-repressedexpression in IRdallsubjects, resulting in high postclamp expression.*, significantdifferencebetweenbracketedgroups(?bonf?0.05).Differentialexpressionofglucosemetabolismgenesinskeletalmuscle.(A18746 ?www.pnas.org?cgi?doi?10.1073?pnas.0903032106 Sears et al.

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Insulin Resistance Is Associated with Elevated Adipocyte Markers in theSkeletal Muscle, Which Are Increased After TZD Treatment. Despite thegross exclusion of perimuscular fat from SM biopsies, SM from IRdsubjectshadhigherlevelsofadipocyte-predominanttranscripts(Data-set S2) compared with SM from NRdsubjects. Several of these genesareinterestingtonote,includingleptin(LEP),adiponectin(ADIPOQ),and retinol-binding protein 4 (RBP4). Leptin was overexpressed ?5-fold in IRdallsubjects at baseline and in IRd?subjects before and afterinsulin-stimulation and/or TZD treatment. Leptin expression in IRd?subjectswasnotsignificantlydifferentfromthatinNRdsubjectsinanycondition.BothADIPOQandRBP4wereoverexpressedinIRdallandIRd?subjects and were significantly further elevated after TZD treat-ment. ADIPOQ and RBP4 were overexpressed in IRd?subjects onlyafter TZD treatment and this pattern was significantly blunted com-pared to the over-expression we observed in IRd?subjects. Leptin,adiponectin,andRBP4areprincipallysecretedbyadipocytes(13)andhave paracrine effects on glucose uptake, fatty acid uptake, and fattyacidoxidationinperipheraltissues(14–17).Thus,alteredexpressionofthesegenessuggeststhattheinsulin-sensitizingeffectsofTZDsinvolveincreased adipocyte-myocyte cross-talk.Genes involved in other aspects of adipocyte biology were differen-tiallyexpressedinSMfromthesubjectgroups.IRdallandIRd?subjectshad higher basal and post-TZD expression of genes that regulate lipiduptake and storage, compared with NRdsubjects. Perilipin (PLIN),fatty acid binding protein (FABP4), stearoyl-CoA desaturase (SCD),cell death-inducing DFFA-like effector c (CIDEC) are among thesegenes. Overexpression of these genes was increased after TZD treat-ment.Overall,theseoverexpressionpatternsweremorepronouncedinIRd?subjects and weakest in IRd?subjects suggesting that elevatedlipid uptake and storage is related to insulin sensitization. In fact,fractional CIDEC expression change correlated with fractional Rdchange (r ? 0.57). We observed significant overexpression of genesinvolved in adipogenesis in SM of IRdsubjects, including CCAAT/enhancer binding protein ? (CEBPA), sterol regulatory element bind-ing transcription factor (SREBF1), and early growth response 2(EGR2). CEBPA overexpression and SREBF1 expression each in-creased significantly after insulin infusion in IRdallsubjects. Interest-ingly, only the IRd?subjects exhibited significant up-regulation ofEGR2 (8.6-fold) after TZD treatment (?Bonf? 0.05). Together, thesefindings suggest that TZD-induced insulin-sensitization is mediated, inpart, by stimulation of intramuscular adipocyte differentiation (seeDiscussion).Fig.3.genesthatareoverexpressedinIRdallsubjectsbeforeTZDtreatment(A)anddown-regulatedinIRd?subjectsafterTZDtreatment(B).(CandD)Schematicsoftheleukocytetransendothelial migration pathway that highlight genes that are overexpressed in IRdallsubjects before TZD treatment (C) and down-regulated in IRd?subjects after TZDtreatment(D).Whiteovals–genesnotdifferentiallyexpressedbetweenNRdandIRdall(AandC)orIRd?(BandD)subjects.Redovals–genessignificantlyoverexpressedbetweenNRdand IRdall(A and C) or IRd?(B and D) subjects. Pink ovals – genes still significantly overexpressed in IRd?subjects compared with NRdsubjects but are significantlydown-regulated compared with pre-TZD levels. Schematics are adapted from KEGG. (E) CD74 was significantly overexpressed in all IRdsubject groups compared with NRdsubjects. After TZD treatment, CD74 expression was significantly down-regulated in IRd?subgroup subjects (pink) but not in IRdallsubjects (red) or IRd?subgroup subjects(yellow).*, significant difference between bracketed groups (?bonf? 0.05). (F) Negative correlation between CD74 expression and Rd (r ? ?0.59). Graph includes pre- andpost-TZDtreatmentdata.IRdallsubjects(pink,red,andyellowtriangles).IRd?subgroupsubjects(pink),IRd?subgroupsubjects(yellow),IRdmidsubgroupsubjects(red).Inflammatorymarkerexpressioninadiposetissueofinsulinresistantsubjects.(AandB)SchematicsoftheantigenprocessingandpresentationpathwayhighlightingSears et al. PNAS ?November 3, 2009 ?vol. 106 ?no. 44 ?18747MEDICAL SCIENCES